Fatimanols Y and Z: two neo-clerodane diterpenoids from Teucrium yemense

Teucrium yemense (Defl.), a medicinal plant, grows in Yemen and Saudi Arabia and is also referred to as Reehal Fatima. The plant has a long history of use in these regions for the treatment of diabetes, rheumatism, and renal conditions. Phytochemical investigation of the aerial parts of T. yemense yielded two previously undescribed neo-clerodane diterpenoids, namely fatimanols Y and Z (1 and 2) along with the known teulepicephin (3), 8-acetylharpagide (4) and teucardosid (5). Structure elucidation was accomplished from their 1D and 2D NMR, ECD, and MS characteristics as well as by comparing them to related reported compounds. The new molecules expand understanding of secondary metabolites of this genus. Compounds 1–5 did not show antimicrobial activity against various bacterial and fungal strains.


Introduction
Teucrium L. (Lamiaceae), commonly known as germanders, is a cosmopolitan genus of about 300 species mainly distributed in South and Central America, Southern Asia, and the Middle East but predominantly prevalent in the Mediterranean basin.Plants in this genus are generally perennial, herbs or shrubs, and the corollas are mostly white to cream-colored with characteristic reduced upper lips. 1,2Teucrium species have been used traditionally as diuretic, diaphoretic, antipyretic, and antiseptic agents for centuries in many parts of the world. 35][6][7] In Egypt, Teucrium is used as an appetizer, expectorant, and hypoglycemic. 8About 300 compounds including avonoids, terpenoids, iridoids, steroids, phenylethanoids and mainly diterpenoids have been reported from different species of Teucrium.The Teucrium genus is a rich source of diterpenoids, particularly neo-clerodanes which are used as chemotaxonomic markers for Teucrium species.More than 220 diterpenes have been described so far from Teucrium. 9eucrium yemense (De.), commonly known as Reehal Fatima, is a therapeutic plant that is frequently grown in Yemen and Saudi Arabia.1][12] Over thirty neo-clerodane diterpene derivatives from this species have been identied and four of them showed potential antidiabetic activity. 2,13,14Based on the aforementioned facts, the aerial parts of T. yemense were selected to explore further chemical investigation.

General experimental procedure
Optical rotations were measured in MeOH using AUTOPOL II Automatic Polarimeter (Rudolph, Hackettstown, NJ, USA).ECD spectra were collected using Olis DSM 20 CD digital spectropolarimeter (Bogart, GA, USA).IR spectra were determined on an Agilent Technologies Cary 630 FTIR.UV spectra were measured on a Thermo Scientic Evolution 201 UV-visible spectrophotometer.NMR experiments were carried out on a Bruker Avance III 400 MHz spectrometer using CD 3 OD as a solvent and methanol residue signals were used as the internal standard.An Agilent Technologies 6200 series mass spectrometer was employed to acquire mass data.Column chromatography (CC) was performed over ash silica gel (Sili-aFlashV ® P60, SiliCycle Inc., USA).Analytical TLC was carried out on silica gel F 254 aluminum sheet (20 cm × 20 cm, SiliCycle, Canada) or reversed phase C-18 aluminum sheet (20 cm × 20 cm, Sorbent Tech., USA).The detection of the spots was made possible by visualization under UV-254 nm and by  spraying with 1% vanillin in H 2 SO 4 -EtOH (10 : 90), followed by heating.Analytical grade solvents (Fischer chemicals) were used for the isolation and purication procedures.

Plant material
Teucrium yemense (De.)aerial parts were collected from Abha, Saudi Arabia in March 2013.The plant identity was conrmed by a taxonomist at the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.The plant material was airdried in shade at room temperature.A voucher specimen, coded Ty/018, was kept in the Pharmacognosy Department, Faculty of Pharmacy, Mansoura University.

Extraction and isolation
The powdered air-dried aerial parts (280 g) were extracted by maceration with aqueous MeOH (90%) at room temperature.The dried extract (30 g), obtained on the removal of the solvent by rotary evaporator, was subjected to vacuum liquid chromatography (VLC) over reversed phase C-18 silica gel (30 cm × 5 cm), eluted rstly with 100% H 2 O, then H 2 O-MeOH 90 3.

In vitro antimicrobial activity
The antimicrobial activity of the isolated compounds was evaluated against Candida albicans, ATCC 90028, Aspergillus fumigatus ATCC 204305, Cryptococcus neoformans ATCC 90113, methicillin-resistant Staphylococcus aureus ATCC 1708 (MRS), Escherichia coli ATCC 2452, Pseudomonas aeruginosa ATCCBAA-2018, Klebsiella pneumonia ATCC 2146 and Enterococcus faecium (VRE) ATCC 700221.From the American Type Culture Collection, the strains were purchased (ATCC, Manassas, VA).An altered version of the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) procedures was used for the susceptibility testing. 19 nal DMSO concentration of 1% was maintained in the assay while serially diluting all samples in 20% DMSO/saline and transferring them in duplicate to 384 well at-bottom microplates.Following the McFarland standard, inocula were created by adjusting the OD630 of microbe suspensions in incubation broth. 20For C. albicans RPMI 1640 (2% dextrose/0.03%glutamine/MOPS at pH 6.0) was used, for C. neoformans, Sabouraud dextrose was used, while, cation-adjusted Mueller-Hinton pH 7.0 for MRS, VRE, E. coli, K. pneumonia, and P. aeruginosa, and RPMI 1640 broth (2% dextrose, 0.03% glutamine, buffered with 0.165 M MOPS at pH 7.0) for A. fumigatus in accordance with the CLSI procedure, to afford recommended inocula as per CLSI protocol.Each assay contained drug controls for bacteria and fungi.MRS, VRE, E. coli, K. pneumoniae, P. aeruginosa, C. albicans, and A. fumigatus were incubated at 35 °C for 48 hours, while C. neoformans was incubated at 35 °C for 68-72 hours.A Bio-Tek plate reader was used to record the optical density (530 nm) or uorescence (544ex/590em) of A. fumigatus, VRE, and MRS before and aer incubation.

Conclusion
Teucrium yemense (De.), known as Reehal Fatima, has lately been identied as a potential source for new neo-clerodane diterpenoids.Consequently, this study's goal was to further explore the chemistry of this plant.The present investigation revealed two undescribed neo-clerodane diterpenoids, namely fatimanol Y and fatimanol Z, together with the known teulepicephin, 8-acetylharpagide and teucardosid from the aerial parts of T. yemense.
),14-dien-20,19-olide and named fatimanol Y. Compound 2 exhibited an [M-H] − ion peak in the HRESIMS at m/z 437.1816 (calcd for C 22 H 29 O 9 , 437.1817) corresponding to the molecular formula of C 22 H 30 O 9 .The NMR data of 2 was comparable to 1 except for the additional resonances [d H/C 2.11/ 21.2 (CH 3 ) and d C 172.5 (carbonyl)] of an acetyl group in 2. The acetyl group was located as an acetoxy group at C-3 based on the HMBC correlations of H-3 (d H 5.28) and methyl group (d H 2.11) with carbonyl (d C 172.5).The complete assignment of 1 H and 13 C NMR resonances was accomplished by HSQC, COSY, and HMBC spectroscopic data.Based on the ECD spectrum (Fig. S35 †), the NOESY correlations (Fig. 3), and characteristic